Clotting and Healing Compositions Containing Keratin Biomaterials

ABSTRACT

Disclosed are optimized keratin preparations for use in medical applications. Methods to produce optimized keratin preparations are provided for use in biomedical applications, particularly for the treatment of bleeding, and for the treatment of wounds. Also disclosed are surgical or paramedic aids comprising a substrate with keratin preparations provided thereon, and kits comprising keratin derivatives packaged in sterile form.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Ser. No. 60/774,442, filed Feb. 17, 2006,U.S. Provisional Patent Application Ser. No. 60/774,587, filed Feb. 17,2006, and U.S. Provisional Patent Application Ser. No. 60/774,920, filedFeb. 17, 2006, the disclosures of each of which is incorporated hereinby reference in its entirety.

This application is related to: Mark E. Van Dyke, U.S. patentapplication Ser. No. 11/205,800, titled: Ambient Stored Blood PlasmaExpanders, filed Aug. 17, 2005; Mark E. Van Dyke, U.S. patentapplication titled: Nerve Regeneration Employing Keratin Biomaterials,filed Feb. 9, 2007 (serial number to be assigned); and Mark E. Van Dyke,PCT Application, titled: Coatings and Biomedical Implants Formed fromKeratin Biomaterials, filed Feb. 16, 2007 (serial number to beassigned).

GOVERNMENT SUPPORT

This invention was made with Government support under contract numberW81XWH-04-1-0105 from the United States Army. The U.S. Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention is generally related to keratin biomaterials andthe use thereof in biomedical applications.

BACKGROUND OF THE INVENTION

Rapid, voluminous hemorrhage instigates a cascade of events that arealmost impossible to reverse without immediate and effectiveintervention. According to the Centers for Disease control, motorvehicle trauma is the leading cause of death for Americans under age 64,with more than 40,000 victims per year (National Vital StatisticsSystem. National Center for Health Statistics. CDC (2003)). The numberof people who die from motor vehicle-related injuries has not changedfor the past 10 years. Historically, more than half of those severelyinjured with concomitant hemorrhage die. While it has been shown thatimmediate intervention is the best method to limit patient mortality(Regel G et al., Acta Anaesthesiol Scand Suppl 1997; 110:71-6), themethods for controlling bleeding in the prehospital setting have notsubstantively changed for centuries (Zimmerman L M and Veith I. Greatideas in the history of surgery. Norman Publishing, San Francisco,Calif. (1993)). Tourniquets and recent innovations in hemostaticpressure dressings have been effective for treatment of surface andextremity wounds, but there are few options for head, neck, chest, andabdominal hemorrhage.

On the battlefield, seventy percent of ballistic injuries result indeath within the first hour. This is due primarily to the massive bloodloss associated with penetrating trauma. In Vietnam, five thousanddeaths resulted from bleeding from the extremities. It was estimatedthat twenty percent of these casualties could have been avoided withbetter first aid (Neel, S., “Medical Support of the U.S. Army in Vietnam1965-1970,” Department of the Army, Washington D.C. (1991)).Surprisingly, the percent of wounded who survive the first hour has notchanged since the U.S. Civil War. In Iraq and Afghanistan, like in allprevious wars, extremity wounds are the predominant injury. Deaths dueto hemorrhage represents the majority of those killed in action in Iraq(Peake J B. N Engl J Med 2005; 352(3):219-22).

Many products exist that effectively treat hemorrhage from theextremities (e.g. tourniquets and hemostatic pressure dressings) as wellas those that work systemically. Numerous large animal preclinical andhuman clinical trials have been conducted and published, and manycomparative investigations of hemostatic agents have been undertaken(Pusateri A E et al., J Trauma 2003; 55(3):518-26; Alam H B et al., JTrauma 2003; 54(6):1077-82; King K et al., Mil Med 2004; 169(9):716-20).Coagulation adjuvants include mineral-based granules (e.g. QuikClot®hemostatic agent) (Turner S A et al., J Biomed Mater Res. 2002;63(1):37-47; Pusateri A E et al., J Trauma 2004; 57(3):555-62; RobinsonK, J Emerg Nurs 2004; 30(2):160-1; Alam H B et al., J Trauma 2004;56(5):974-83), numerous dressings (Rothwell S W et al., Thromb Res 2003;108(5-6);335-40; Alencar de Queiroz A A et al., J Biomed Mat Res A 2003;64(1):147-54; Vournakis J N et al., J Surg Res 2003; 113(1):1-5;Connolly R J, J Trauma 2004; 57(1 Suppl):S26-8; King D R et al., JTrauma 2004; 57(4):756-9), clotting factors (Martinowitz U et al., JTrauma 2001; 50(4):721-9; Schreiber M A et al., J Trauma 2002;53(2):252-9), and surgical approaches (Jaskille A et al., J Trauma 2005;59(6):1305-8; Takasu A et al., J Trauma 2004; 56(5):984-90). Resultshave been mixed and are dependent on the model used. The zeolite-basedQuikClot® hemostatic agent material has done well in some large animaltrials, as has the chitosan-based bandage, HemCon® hemostatic bandage.In one extensive comparative trial, fibrin dressings were clearlysuperior (Pusateri A E et al., J Trauma 2003; 55(3):518-26). However,each of these hemostats has its challenges and limitations. QuikClot®hemostatic agent is known to produce localized heating which has beenshown to damage tissue. Both HemCon® hemostatic bandage and QuikClot®hemostatic agent are not intended for internal injuries and must beremoved from the wound site, and fibrin bandages are expensive and notyet approved by the Food and Drug Administration.

None of these materials, with the exception of a fibrin foam productthat is still in development, can be applied without clear access to thesite of hemorrhage, a major goal of first responders. Therefore thereremains a great need for optimal materials that can control serioushemorrhage and promote wound healing.

The use of materials derived from keratin in medicine is not new. Theearliest documented use of keratin in medicine comes from a Chineseherbalist named Li Shi-Zhen (Ben Cao Gang Mu. Materia Medica, adictionary of Chinese herbs, written by Li Shi Zhen (1518-1593)). Over a38-year period, he wrote a collection of 800 books known as the Ben CaoGang Mu. These books were published in 1596, three years after hisdeath. Among the more than 11,000 prescriptions described in thesevolumes, is a substance known as Xue Yu Tan, also known as CrinisCarbonisatus, that is made up of ground ash from pyrolized human hair.The stated indications for Xue Yu Tan were accelerated wound healing andblood clotting.

In the early 1800s, when proteins were still being called albuminoids(albumin was a well known protein at that time), many different kinds ofproteins were being discovered. Around 1849, the word “keratin” appearsin the literature to describe the material that made up hard tissuessuch as animal horns and hooves (keratin comes from the Greek “kera”meaning horn). This new protein intrigued scientists because it did notbehave like other proteins. For example, the normal methods used fordissolving proteins were ineffective with keratin. Although methods suchas burning and grinding had been known for some time, many scientistsand inventors were more interested in dissolving hair and horns in orderto make better products.

During the years from 1905 to 1935, many methods were developed toextract keratins using oxidative and reductive chemistries (Breinl F andBaudisch O, Z physiol Chem 1907; 52:158-69; Neuberg C, U.S. Pat. No.926,999, Jul. 6, 1909; Lissizin T, Biochem Bull 1915; 4:18-23; Zdenko S,Z physiol Chem 1924; 136:160-72; Lissizin T, Z physiol Chem 1928;173:309-11). By the late 1920s many techniques had been developed forbreaking down the structures of hair, horns, and hooves, but scientistswere confused by the behavior of some of these purified proteins.Scientists soon concluded that many different forms of keratin werepresent in these extracts, and that the hair fiber must be a complexstructure, not simply a strand of protein. In 1934, a key research paperwas published that described different types of keratins, distinguishedprimarily by having different molecular weights (Goddard D R andMichaelis L, J Biol Chem 1934; 106:605-14). This seminal paperdemonstrated that there were many different keratin homologs, and thateach played a different role in the structure and function of the hairfollicle.

Earlier work at the University of Leeds and the Wool Industries ResearchAssociation in the United Kingdom had shown that wool and other fiberswere made up of an outer cuticle and a central cortex. Building on thisinformation, scientists at CSIRO conducted many of the most fundamentalstudies on the structure and composition of wool. Using X-raydiffraction and electron microscopy, combined with oxidative andreductive chemical methods, CSIRO produced the first complete diagram ofa hair fiber (Rivett D E et al., “Keratin and Wool Research,” The LennoxLegacy, CSIRO Publishing; Collingwood, VIC, Australia; 1996).

In 1965, CSIRO scientist W. Gordon Crewther and his colleagues publishedthe definitive text on the chemistry of keratins (Crewther W G et al.,The Chemistry of Keratins. Anfinsen C B Jr et al., editors. Advances inProtein Chemistry 1965. Academic Press. New York:191-346). This chapterin Advances in Protein Chemistry contained references to more than 640published studies on keratins. Once scientists knew how to extractkeratins from hair fibers, purify and characterize them, the number ofderivative materials that could be produced with keratins grewexponentially. In the decade beginning in 1970, methods to formextracted keratins into powders, films, gels, coatings, fibers, andfoams were being developed and published by several research groupsthroughout the world (Anker C A, U.S. Pat. No. 3,642,498, Feb. 15, 1972;Kawano Y and Okamoto S, Kagaku To Seibutsu 1975; 13(5):291-223; OkamotoS, Nippon Shokuhin Kogyo Gakkaishi 1977; 24(1):40-50). All of thesemethods made use of the oxidative and reductive chemistries developeddecades earlier.

In 1982, Japanese scientists published the first study describing theuse of a keratin coating on vascular grafts as a way to eliminate bloodclotting (Noishiki Y et al., Kobunshi Ronbunshu 1982; 39(4):221-7), aswell as experiments on the biocompatibility of keratins (Ito H et al.,Kobunshi Ronbunshu 1982; 39(4):249-56). Soon thereafter in 1985, tworesearchers from the UK published a review article speculating on theprospect of using keratin as the building block for new biomaterialsdevelopment (Jarman T and Light J, World Biotech Rep 1985; 1:505-12). In1992, the development and testing of a host of keratin-basedbiomaterials was the subject of a doctoral thesis for French graduatestudent Isabelle Valherie (Valherie I and Gagnieu C. Chemicalmodifications of keratins: Preparation of biomaterials and study oftheir physical, physiochemical and biological properties. Doctoralthesis. Inst Natl Sci Appl Lyon, France 1992). Soon thereafter, Japanesescientists published a commentary in 1993 on the prominent positionkeratins could take at the forefront of biomaterials development(Various Authors, Kogyo Zairyo 1993; 41(15) Special issue 2:106-9).

Taken together, the aforementioned body of published work isillustrative of the unique chemical, physical, and biological propertiesof keratins. However, there remains a great need for optimized keratinpreparations for use in biomedical applications, particularly for thetreatment of bleeding, and for the treatment of wounds.

SUMMARY OF THE INVENTION

An aspect of the present invention is a pharmaceutical compositioncomprising a keratin derivative (e.g., keratose, kerateine, or acombination thereof) and optionally, at least one additional activeingredient.

Another aspect of the present invention is a method for treatingbleeding in a subject afflicted with a bleeding wound comprisingapplying a positively charged composition to said wound in an amounteffective to treat said bleeding. In some embodiments, said positivelycharged composition comprises, consists or consists essentially ofderivatives of keratin, collagen, mucin, elastin, gelatin, fibronectin,vitronectin and laminin.

A further aspect of the present invention is a method for treatingbleeding in a subject afflicted with a bleeding wound, comprising:applying a keratin derivative to the wound in an amount effective totreat the bleeding. In some embodiments, the keratin derivativecomprises, consists of or consists essentially of alpha keratose, gammakeratose, acidic alpha keratose, basic alpha keratose, acidic gammakeratose, basic gamma keratose, alpha kerateine, gamma kerateine, acidicalpha kerateine, basic alpha kerateine, acidic gamma kerateine, basicgamma kerateine, or combinations thereof.

Yet another aspect of the present invention is a method of treating awound in a subject in need thereof, comprising: topically applying akeratin derivative to the wound in an amount effective to treat thewound. In some embodiments the keratin derivative comprises, consists ofor consists essentially of alpha keratose, gamma keratose, acidic alphakeratose, basic alpha keratose, acidic gamma keratose, basic gammakeratose, alpha kerateine, gamma kerateine, acidic alpha kerateine,basic alpha kerateine, acidic gamma kerateine, basic gamma kerateine, orcombinations thereof.

Another aspect of the present invention is a surgical or paramedic aid,comprising: a solid, physiologically acceptable substrate; and a keratinderivative on the substrate. In some embodiments the keratin derivativecomprises, consists of or consists essentially of alpha keratose, gammakeratose, acidic alpha keratose, basic alpha keratose, acidic gammakeratose, basic gamma keratose, alpha kerateine, gamma kerateine, acidicalpha kerateine, basic alpha kerateine, acidic gamma kerateine, basicgamma kerateine, or combinations thereof.

A still further aspect of the present invention is a kit comprising akeratin derivative and a container in which said keratin derivative ispackaged in sterile form. In some embodiments the keratin derivativecomprises, consists of or consists essentially of alpha keratose, gammakeratose, acidic alpha keratose, basic alpha keratose, acidic gammakeratose, basic gamma keratose, alpha kerateine, gamma kerateine, acidicalpha kerateine, basic alpha kerateine, acidic gamma kerateine, basicgamma kerateine, or combinations thereof.

Another aspect of the present invention is the use of a keratinderivative as described herein for the preparation of a composition ormedicament for carrying out a method of treatment as described herein,or for making an article of manufacture as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Kaplan-Mayer Survival Graph: Time is presented in minutes on alogarithmic scale. All animals in the control group died within 60minutes. One animal from the keratin and one from the HemCon® hemostaticbandage group was sacrificed upon recommendation of the animal carestaff Overall, keratin outperformed the other groups with only one deathcompared with 2 deaths in the QuikClot® hemostatic agent and HemCon®hemostatic bandage groups.

FIG. 2. Shed Blood: Blood loss is normalized to body weight andexpressed as percentage of body weight. Keratin and QuikClot® hemostaticagent groups lost significantly (*) less blood than the control andHemCon® hemostatic bandage groups.

FIG. 3. Mean Arterial Pressure (MAP): Blood pressure is expressed inpercentage of initial pressure. The negative control and QuikClot®hemostatic agent groups showed a steep drop in pressure to 40% ofinitial MAP. Animals treated with keratin or HemCon® hemostatic bandagewere able to stabilize the MAP around 80% of initial pressure. Thesedifferences were not statistically different compared to the controlgroup.

FIG. 4. Shock Index (SI): The modified shock index was calculated bydividing heart rate by MAP. This index is clinically used to assess theseverity of a shock, with low values being better. The animals in thekeratin group showed compensated low values over the entire studyperiod, while QuikClot® hemostatic agent and HemCon® hemostatic bandagegroups had similar values as the negative control. There was nostatistical significance between the groups.

FIG. 5. Histological Assessment: Representative tissue sections stainedwith hematoxylin and eosin, 50×. A) The negative control group showssigns of poor perfusion with wide and empty sinusoids. The surface islacking a functional blood clot. B) The surface of the QuikClot®hemostatic agent treated samples shows an area of necrosis (arrow) andclotting. Only minimal cellular infiltration and tissue regeneration isvisible. The void areas represent the removed QuikClot® hemostatic agentgranules. C) Tissue samples from HemCon® hemostatic bandage treatedanimals showed patchy areas of adherent clotted blood, where there was alow level of cellular infiltration. D) Liver samples from animalstreated with keratin show a thick layer of keratin biomaterial attachedto the injured surface. There are signs of excellent biocompatibilitywith a high cellular activity and the formation of early granular tissue(large arrow) in the spaces between the keratin. Further, there is ahigh level of direct contact of hepatocytes with the keratin biomaterial(small arrow).

FIG. 6: Keratin Treated Group, High Magnification: A) Formation of earlygranulation-like tissue within the spaces of the keratin gel, 200×. B)Interface between keratin gel and liver tissue showing integration ofthe biomaterial and tissue, and early cellular infiltration, 400×.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The disclosures of all United States patent references cited herein areto be incorporated herein by reference.

“Subjects” (or “patients”) to be treated with the methods andcompositions described herein include both human subjects and animalsubjects (particularly other mammalian subjects such as dogs, cats,horses, monkeys, etc.) for veterinary purposes. Human subjects areparticularly preferred. The subjects may be male or female and may beany age, including neonate, infant, juvenile, adolescent, adult, andgeriatric subjects.

The ability of extracted keratin solutions to spontaneouslyself-assemble at the micron scale was published in two papers in 1986and 1987 (Thomas H et al., Int J Biol Macromol 1986; 8:258-64; van deLöcht M, Melliand Textilberichte 1987; 10:780-6). This phenomenon is notsurprising given the highly controlled superstructure whence hairkeratins are obtained. When processed correctly, this ability toself-assemble can be preserved and used to create regular architectureson a size scale conducive to cellular infiltration. When keratins arehydrolyzed (e.g., with acids or bases), their molecular weight isreduced and they lose the ability to self-assemble. Therefore,processing conditions that minimize hydrolysis are preferred.

This ability to self-assemble is a particularly useful characteristicfor tissue engineering scaffolds for two reasons. First, self-assemblyresults in a highly regular structure with reproducible architectures,dimensionality, and porosity. Second, the fact that these architecturesform of their own accord under benign conditions allows for theincorporation of cells as the matrix is formed. These two features arecritically important to any system that attempts to mimic the nativeextracellular matrix (ECM).

Cellular recognition is also an important characteristic of biomaterialsthat seek to mimic the ECM. Such recognition is facilitated by thebinding of cell surface integrins to specific amino acid motifspresented by the constituent ECM proteins. Predominant proteins includecollagen and fibronectin, both of which have been extensively studiedwith regard to cell binding. Both proteins contain several regions thatsupport attachment by a wide variety of cell types. It has been shownthat in addition to the widely know Arginine-Glycine-Aspartic Acid (RGD)motif, the “X”-Aspartic Acid-“Y” motif on fibronectin is also recognizedby the integrin α4β1, where X equals Glycine, Leucine, or Glutamic Acid,and Y equals Serine or Valine. Keratin biomaterials derived from humanhair contain these same binding motifs. A search of the NCBI proteindatabase revealed sequences for 71 discrete, unique human hair keratinproteins. Of these, 55 are from the high molecular weight, low sulfur,alpha-helical family. This group of proteins is often referred to as thealpha-keratins and is responsible for imparting toughness to human hairfibers. These alpha-keratins have molecular weights greater than 40 kDaand an average cysteine (the main amino acid responsible for inter- andintramolecular protein bonding) content of 4.8 mole percent. Moreover,analysis of the amino acid sequences of these alpha keratin proteinsshowed that 78% contain at least one fibronectin-like integrin receptorbinding motif, and 25% contain at least two or more. Two recent papershave highlighted the fact that these binding sites are likely present onthe surface of keratin biomaterials by demonstrating excellent celladhesion onto processed keratin foams (Tachibana A et al., J Biotech2002; 93:165-70; Tachibana A et al., Biomaterials 2005; 26(3):297-302).

Other examples of natural polymers that may be utilized in a similarfashion to the disclosed keratin preparations include, but are notlimited to, collagen, gelatin, fibronectin, vitronectin, laminin,fibrin, mucin, elastin, nidogen (entactin), proteoglycans, etc. (See,e.g., U.S. Pat. No. 5,691,203 to Katsuen et al.).

There are two theories for the biological activity of human hairextracts. The first is that the human hair keratins (“HHKs”) themselvesare biologically active. Over 70 human hair keratins are known and theircDNA-derived sequences published. However, the full compliment of HHKsis unknown and estimates of over 100 have been proposed (Gillespie J M,The structural proteins of hair: isolation characterization, andregulation of biosynthesis. Goldsmith L A (editor), Biochemistry andphysiology of the skin (1983), Oxford University Press. New York;475-510). Within the complete range of HHKs are a small number that havebeen shown to participate in wound contracture and cell migration(Martin, P, Science 1997; 276:75-81). In particular, keratins K-6 andK-16 are expressed in the epidermis during wound healing and are alsofound in the outer root sheath of the hair follicle (Bowden P E,Molecular Aspects of Dermatology (1993), John Wiley & Sons, Inc.,Chichester:19-54). The presence of these HHKs in extracts of human hair,and their subsequent dosing directly into a wound bed, may beresponsible for “shortcutting” the otherwise lengthy process ofdifferentiation, migration, and proliferation, or for alleviating somebiochemical deficiency, thereby accelerating the tissue repair andregeneration process.

It has been known for more than a decade that growth factors such asbone morphogenetic protein-4 (BMP-4) and other members of thetransforming growth factor-β (TGF-β) superfamily are present indeveloping hair follicles (Jones C M et al., Development 1991;111:531-42; Lyons K M et al., Development 1990; 109:833-44; Blessings Met al., Genes and Develop 1993; 7:204-15). In fact, more than 30 growthfactors and cytokines are involved in the growth of a cycling hairfollicle (Hardy M H, Trends Genet 1992; 8(2):55-61; Stenn K S et al., JDermato Sci 1994; 7S:S109-24; Rogers G E, Int J Dev Biol 2004;48(2-3):163-70). Many of these molecules have a pivotal role in theregeneration of a variety of tissues. It is highly probable that anumber of growth factors become entrained within human hair whencytokines bind to stem cells residing in the bulge region of the hairfollicle (Panteleyev A A et al., J Cell Sci 2001; 114:3419-31). Thesegrowth factors would most certainly be extracted along with the keratinsfrom end-cut human hair. This observation is not without precedent, asit has previously been shown that many different types of growth factorsare present in the extracts of various tissues, and that their activityis maintained even after chemical extraction. Observations such as theseshow mounting evidence that a number of growth factors may be present inend-cut human hair, and that the keratins may be acting as a highlyeffective delivery matrix of, inter alia, these growth factors.

Keratins are a family of proteins found in the hair, skin, and othertissues of vertebrates. Hair is a unique source of human keratinsbecause it is one of the few human tissues that is readily available andinexpensive. Although other sources of keratins are acceptablefeedstocks for the present invention, (e.g. wool, fur, horns, hooves,beaks, feathers, scales, and the like), human hair is preferred for usewith human subjects because of its biocompatibility.

Keratins can be extracted from human hair fibers by oxidation orreduction using methods that have been published in the art (See, e.g.,Crewther W G et al. The chemistry of keratins, in Advances in ProteinChemistry 1965; 20:191-346). These methods typically employ a two-stepprocess whereby the crosslinked structure of keratins is broken down byeither oxidation or reduction. In these reactions, the disulfide bondsin cysteine amino acid residues are cleaved, rendering the keratinssoluble (Scheme 1). The cuticle is essentially unaffected by thistreatment, so the majority of the keratins remain trapped within thecuticle's protective structure. In order to extract these keratins, asecond step using a denaturing solution must be employed. Alternatively,in the case of reduction reactions, these steps can be combined.Denaturing solutions known in the art include urea, transition metalhydroxides, surfactant solutions, and combinations thereof. Preferredmethods use aqueous solutions of tris in concentrations between 0.1 and1.0 M, and urea solutions between 0.1 and 10M, for oxidation andreduction reactions, respectively.

If one employs an oxidative treatment, the resulting keratins arereferred to as “keratoses.” If a reductive treatment is used, theresulting keratins are referred to as “kerateines” (See Scheme 1)

Crude extracts of keratins, regardless of redox state, can be furtherrefined into “gamma” and “alpha” fractions, e.g., by isoelectricprecipitation. High molecular weight keratins, or “alpha keratins,”(alpha helical), are thought to derive from the microfibrillar regionsof the hair follicle, and typically range in molecular weight from about40-85 kiloDaltons. Low molecular weight keratins, or “gamma keratins,”(globular), are thought to derive from the extracellular matrix regionsof the hair follicle, and typically range in molecular weight from about10-15 kiloDaltons. (See Crewther W G et al. The chemistry of keratins,in Advances in Protein Chemistry 1965; 20:191-346)

Even though alpha and gamma keratins possess unique properties, theproperties of subfamilies of both alpha and gamma keratins can only berevealed through more sophisticated means of purification. For example,keratins may be fractionated into “acidic” and “basic” proteinfractions. A preferred method of fractionation is ion exchangechromatography. These fractions possess unique properties, such as theirdifferential effects on blood cell aggregation (See Table 1 below; Seealso: U.S. Patent Application Publication No. 2006/0051732).

“Keratin derivative” as used herein refers to any keratin fractionation,derivative or mixture thereof, alone or in combination with otherkeratin derivatives or other ingredients, including but not limited toalpha keratose, gamma keratose, alpha kerateine, gamma kerateine, metakeratin, keratin intermediate filaments, and combinations thereof,including the acidic and basic constituents thereof unless specifiedotherwise, along with variations thereof that will be apparent topersons skilled in the art in view of the present disclosure. In someembodiments, the keratin derivative comprises, consists or consistsessentially of a particular fraction or subfraction of keratin. Thederivative may comprise, consist or consist essentially of at least 80,90, 95 or 99 percent by weight of said fraction or subfraction (ormore).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic alpha keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha keratose, where the alpha keratosecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of acidic alpha keratose (or more), and where thealpha keratose comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of basic alpha keratose (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of basic alpha keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha keratose, where the alpha keratosecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of basic alpha keratose (or more), and where thealpha keratose comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of acidic alpha keratose (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic alpha kerateine.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha kerateine, where the alpha kerateinecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of acidic alpha kerateine (or more), and where thealpha kerateine comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of basic alpha kerateine (orless).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of basic alpha kerateine.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of alpha kerateine, where the alpha kerateinecomprises, consists of or consists essentially of at least 80, 90, 95 or99 percent by weight of basic alpha kerateine (or more), and where thealpha kerateine comprises, consists of or consists essentially of notmore than 20, 10, 5 or 1 percent by weight of acidic alpha kerateine (orless).

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated alpha+gamma-kerateines. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic alpha+gamma-kerateines. In some embodiments, thekeratin derivative comprises, consists of or consists essentially ofbasic alpha+gamma-kerateines.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated alpha+gamma-keratose. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic alpha+gamma-keratose. In some embodiments, thekeratin derivative comprises, consists of or consists essentially ofbasic alpha+gamma-keratose.

In some embodiments, the keratin derivative comprises, consists of orconsists essentially of unfractionated beta-keratose (e.g., derived fromcuticle). In some embodiments, the keratin derivative comprises,consists of or consists essentially of basic beta-keratose. In someembodiments, the keratin derivative comprises, consists of or consistsessentially of acidic beta-keratose.

The basic alpha keratose is preferably produced by separating basicalpha keratose from a mixture comprising acidic and basic alphakeratose, e.g., by ion exchange chromatography, and optionally the basicalpha keratose has an average molecular weight of from 10 to 100 or 200kiloDaltons. More preferably, the average molecular weight is from 30 or40 to 90 or 100 kiloDaltons. Optionally but preferably the processfurther comprises the steps of re-dissolving said basic alpha-keratosein a denaturing and/or buffering solution, optionally in the presence ofa chelating agent to complex trace metals, and then re-precipitating thebasic alpha keratose from the denaturing solution. It will beappreciated that the composition preferably contains not more than 5, 2,1, or 0.1 percent by weight of acidic alpha keratose, or less.

The acidic alpha keratose is preferably produced by a reciprocal of theforegoing technique: that is, by separating and retaining acidic alphakeratose from a mixture of acidic and basic alpha keratose, e.g., by ionexchange chromatography, and optionally the acidic alpha keratose has anaverage molecular weight of from 10 to 100 or 200 kiloDaltons. Morepreferably, the average molecular weight is from 30 or 40 to 90 or 100kiloDaltons. Optionally but preferably the process further comprises thesteps of re-dissolving said acidic alpha-keratose in a denaturingsolution and/or buffering solution, optionally in the presence of achelating agent to complex trace metals, and then re-precipitating thebasic alpha keratose from the denaturing solution. It will beappreciated that the composition preferably contains not more than 5, 2,1, or 0.1 percent by weight of basic alpha keratose, or less.

Basic and acidic fractions of other keratoses can be prepared in likemanner as described above for basic and acidic alpha keratose.

The basic alpha kerateine is preferably produced by separating basicalpha kerateine from a mixture of acidic and basic alpha kerateine,e.g., by ion exchange chromatography, and optionally the basic alphakerateine has an average molecular weight of from 10 to 100 or 200kiloDaltons. Optionally but preferably the process further comprises thesteps of re-dissolving said basic alpha-kerateine in a denaturing and/orbuffering solution, optionally in the presence of a chelating agent tocomplex trace metals, and then re-precipitating the basic alphakerateine from the denaturing solution. It will be appreciated that thecomposition preferably contains not more than 5, 2, 1, or 0.1 percent byweight of acidic alpha kerateine, or less.

The acidic alpha kerateine is preferably produced by a reciprocal of theforegoing technique: that is, by separating and retaining acidic alphakerateine from a mixture of acidic and basic alpha kerateine, e.g., byion exchange chromatography, and optionally the acidic alpha kerateinehas an average molecular weight of from 10 to 100 or 200 kiloDaltons.More preferably, the average molecular weight is from 30 or 40 to 90 or100 kiloDaltons. Optionally but preferably the process further comprisesthe steps of re-dissolving said acidic alpha-kerateine in a denaturingand/or buffering solution), optionally in the presence of a chelatingagent to complex trace metals, and then re-precipitating the basic alphakerateine from the denaturing solution. It will be appreciated that thecomposition preferably contains not more than 5, 2, 1, or 0.1 percent byweight of basic alpha kerateine, or less.

Basic and acidic fractions of other kerateines can be prepared in likemanner as described above for basic and acidic alpha kerateine.

Keratin materials are derived from any suitable source including, butnot limited to, wool and human hair. In one embodiment keratin isderived from end-cut human hair, obtained from barbershops and salons.The material is washed in hot water and mild detergent, dried, andextracted with a nonpolar organic solvent (typically hexane or ether) toremove residual oil prior to use.

Keratoses. Keratose fractions are obtained by any suitable technique. Inone embodiment they are obtained using the method of Alexander andcoworkers (P. Alexander et al., Biochem. J. 46, 27-32 (1950)).Basically, the hair is reacted with an aqueous solution of peraceticacid at concentrations of less than ten percent at room temperature for24 hours. The solution is filtered and the alpha-keratose fractionprecipitated by addition of mineral acid to a pH of approximately 4. Thealpha-keratose is separated by filtration, washed with additional acid,followed by dehydration with alcohol, and then freeze dried. Increasedpurity can be achieved by redissolving the keratose in a denaturingsolution such as 7M urea, aqueous ammonium hydroxide solution, or 20 mMtris base buffer solution (e.g., Trizma® base), re-precipitating,re-dissolving, dialyzing against deionized water, and re-precipitatingat pH 4.

A preferred method for the production of keratoses is by oxidation withhydrogen peroxide, peracetic acid, or performic acid. A most preferredoxidant is peracetic acid. Preferred concentrations range from 1 to 10weight/volume percent (w/v %), the most preferred being approximately 2w/v %. Those skilled in the art will recognize that slight modificationsto the concentration can be made to effect varying degrees of oxidation,with concomitant alterations in reaction time, temperature, and liquidto solid ratio. It has also been discussed by Crewther et al. thatperformic acid offers the advantage of minimal peptide bond cleavagecompared to peracetic acid. However, peractic acid offers the advantagesof cost and availability. A preferred oxidation temperature is between 0and 100 degrees Celsius (° C.). A most preferred oxidation temperatureis 37° C. A preferred oxidation time is between 0.5 and 24 hours. A mostpreferred oxidation time is 12 hours. A preferred liquid to solid ratiois from 5 to 100:1. A most preferred ratio is 20:1. After oxidation, thehair is rinsed free of residual oxidant using a copious amount ofdistilled water.

The keratoses can be extracted from the oxidized hair using an aqueoussolution of a denaturing agent. Protein denaturants are well known inthe art, but preferred solutions include urea, transition metalhydroxides (e.g. sodium and potassium hydroxide), ammonium hydroxide,and tris(hydroxymethyl)aminomethane (tris base). A preferred solution isTrizma® base (a brand of tris base) in the concentration range from 0.01to 1M. A most preferred concentration is 0.1M. Those skilled in the artwill recognize that slight modifications to the concentration can bemade to effect varying degrees of extraction, with concomitantalterations in reaction time, temperature, and liquid to solid ratio. Apreferred extraction temperature is between 0 and 100 degrees Celsius. Amost preferred extraction temperature is 37° C. A preferred extractiontime is between 0.5 and 24 hours. A most preferred extraction time is 3hours. A preferred liquid to solid ratio is from 5 to 100:1. A mostpreferred ratio is 40:1. Additional yield can be achieved withsubsequent extractions with dilute solutions of tris base or deionized(DI) water. After extraction, the residual solids are removed fromsolution by centrifugation and/or filtration.

The crude extract can be isolated by first neutralizing the solution toa pH between 7.0 and 7.4. A most preferred pH is 7.4. Residualdenaturing agent is removed by dialysis against DI water. Concentrationof the dialysis retentate is followed by lyophilization or spray drying,resulting in a dry powder mixture of both gamma- and alpha-keratose.Alternately, alpha-keratose is isolated from the extract solution bydropwise addition of acid until the pH of the solution reachesapproximately 4.2. Preferred acids include sulfuric, hydrochloric, andacetic. A most preferred acid is concentrated hydrochloric acid.Precipitation of the alpha fraction begins at around pH 6.0 andcontinues until approximately 4.2. Fractional precipitation can beutilized to isolate different ranges of protein with differentisoelectric properties. Solid alpha-keratose can be recovered bycentrifugation or filtration.

The alpha keratose can be further purified by re-dissolving the solidsin a denaturing solution. The same denaturing solutions as thoseutilized for extraction can be used, however a preferred denaturingsolution is tris base. Ethylene diamine tetraacetic acid (EDTA) can beadded to complex and remove trace metals found in the hair. A preferreddenaturing solution is 20 mM tris base with 20 mM EDTA or DI water with20 mM EDTA. If the presence of trace metals is not detrimental to theintended application, the EDTA can be omitted. The alpha-keratose isre-precipitated from this solution by dropwise addition of hydrochloricacid to a final pH of approximately 4.2. Isolation of the solid is bycentrifugation or filtration. This process can be repeated several timesto further purify the alpha-keratose.

The gamma keratose fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and freezedried. Increased purity can be achieved by redissolving the keratose ina denaturing solution such as 7M urea, aqueous ammonium hydroxidesolution, or 20 mM tris buffer solution, reducing the pH to 4 byaddition of a mineral acid, removing any solids that form, neutralizingthe supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratose solution bydistillation.

After removal of the alpha keratose, the concentration of gamma keratosefrom a typical extraction solution is approximately 1-2%. The gammakeratose fraction can be isolated by addition to a water-misciblenon-solvent. To effect precipitation, the gamma-keratose solution can beconcentrated by evaporation of excess water. This solution can beconcentrated to approximately 10-20% by removal of 90% of the water.This can be done using vacuum distillation or by falling filmevaporation. After concentration, the gamma-keratose solution is addeddropwise to an excess of cold non-solvent. Suitable non-solvents includeethanol, methanol, acetone, and the like. A most preferred non-solventis ethanol. A most preferred method is to concentrate the gamma keratosesolution to approximately 10 w/v % protein and add it dropwise to an8-fold excess of cold ethanol. The precipitated gamma keratose can beisolated by centrifugation or filtration and dried. Suitable methods fordrying include freeze drying (lyophilization), air drying, vacuumdrying, or spray drying. A most preferred method is freeze drying.

Kerateines. Kerateine fractions can be obtained using a combination ofthe methods of Bradbury and Chapman (J. Bradbury et al., Aust. J. Biol.Sci. 17, 960-72 (1964)) and Goddard and Michaelis (D. Goddard et al., J.Biol. Chem. 106, 605-14 (1934)). Essentially, the cuticle of the hairfibers is removed ultrasonically in order to avoid excessive hydrolysisand allow efficient reduction of cortical disulfide bonds in a secondstep. The hair is placed in a solution of dichloroacetic acid andsubjected to treatment with an ultrasonic probe. Further refinements ofthis method indicate that conditions using 80% dichloroacetic acid,solid to liquid of 1:16, and an ultrasonic power of 180 Watts areoptimal (H. Ando et al., Sen'i Gakkaishi 31(3), T81-85 (1975)). Solidfragments are removed from solution by filtration, rinsed and air dried,followed by sieving to isolate the hair fibers from removed cuticlecells.

In some embodiments, following ultrasonic removal of the cuticle, alpha-and gamma kerateines are obtained by reaction of the denuded fibers withmercaptoethanol. Specifically, a low hydrolysis method is used at acidicpH (E. Thompson et al., Aust. J. Biol. Sci. 15, 757-68 (1962)). In atypical reaction, hair is extracted for 24 hours with 4M mercaptoethanolthat has been adjusted to pH 5 by addition of a small amount ofpotassium hydroxide in deoxygenated water containing 0.02M acetatebuffer and 0.001M surfactant.

The solution is filtered and the alpha kerateine fraction precipitatedby addition of mineral acid to a pH of approximately 4. The alphakerateine is separated by filtration, washed with additional acid,followed by dehydration with alcohol, and then dried under vacuum.Increased purity is achieved by re-dissolving the kerateine in adenaturing solution such as 7M urea, aqueous ammonium hydroxidesolution, or 20 mM tris buffer solution, re-precipitating,re-dissolving, dialyzing against deionized water, and re-precipitatingat pH 4.

The gamma kerateine fraction remains in solution at pH 4 and is isolatedby addition to a water-miscible organic solvent such as alcohol,followed by filtration, dehydrated with additional alcohol, and driedunder vacuum. Increased purity can be achieved by redissolving thekerateine in a denaturing solution such as 7M urea, aqueous ammoniumhydroxide solution, or 20 mM tris buffer solution, reducing the pH to 4by addition of a mineral acid, removing any solids that form,neutralizing the supernatant, re-precipitating the protein with alcohol,re-dissolving, dialyzing against deionized water, and reprecipitating byaddition to alcohol. The amount of alcohol consumed in these steps canbe minimized by first concentrating the keratin solution bydistillation.

In an alternate method, the kerateine fractions are obtained by reactingthe hair with an aqueous solution of sodium thioglycolate.

A preferred method for the production of kerateines is by reduction ofthe hair with thioglycolic acid or beta-mercaptoethanol. A mostpreferred reductant is thioglycolic acid (TGA). Preferred concentrationsrange from 1 to 10M, the most preferred being approximately 1.0M. Thoseskilled in the art will recognize that slight modifications to theconcentration can be made to effect varying degrees of reduction, withconcomitant alterations in pH, reaction time, temperature, and liquid tosolid ratio. A preferred pH is between 9 and 11. A most preferred pH is10.2. The pH of the reduction solution is altered by addition of base.Preferred bases include transition metal hydroxides, sodium hydroxide,and ammonium hydroxide. A most preferred base is sodium hydroxide. ThepH adjustment is effected by dropwise addition of a saturated solutionof sodium hydroxide in water to the reductant solution. A preferredreduction temperature is between 0 and 100° C. A most preferredreduction temperature is 37° C. A preferred reduction time is between0.5 and 24 hours. A most preferred reduction time is 12 hours. Apreferred liquid to solid ratio is from 5 to 100:1. A most preferredratio is 20:1. Unlike the previously described oxidation reaction,reduction is carried out at basic pH. That being the case, keratins arehighly soluble in the reduction media and are expected to be extracted.The reduction solution is therefore combined with the subsequentextraction solutions and processed accordingly.

Reduced keratins are not as hydrophilic as their oxidized counterparts.As such, reduced hair fibers will not swell and split open as willoxidized hair, resulting in relatively lower yields. Another factoraffecting the kinetics of the reduction/extraction process is therelative solubility of kerateines. The relative solubility rankings inwater is gamma-keratose>alpha-keratose>gamma-kerateine>alpha-kerateinefrom most to least soluble. Consequently, extraction yields from reducedhair fibers are not as high. This being the case, subsequent extractionsare conducted with additional reductant plus denaturing agent solutions.Preferred solutions for subsequent extractions include TGA plus urea,TGA plus tris base, or TGA plus sodium hydroxide. After extraction,crude fractions of alpha- and gamma-kerateine can be isolated using theprocedures described for keratoses. However, precipitates of gamma- andalpha-kerateine re-form their cystine crosslinks upon exposure tooxygen. Precipitates must therefore be re-dissolved quickly to avoidinsolubility during the purification stages, or precipitated in theabsence of oxygen.

Residual reductant and denaturing agents can be removed from solution bydialysis. Typical dialysis conditions are 1 to 2% solution of kerateinesdialyzed against DI water for 24 to 72 hours. Those skilled in the artwill recognize that other methods exist for the removal of low molecularweight contaminants in addition to dialysis (e.g. microfiltration,chromatography, and the like). The use of tris base is only required forinitial solubilization of the kerateines. Once dissolved, the kerateinesare stable in solution without the denaturing agent. Therefore, thedenaturing agent can be removed without the resultant precipitation ofkerateines, so long as the pH remains at or above neutrality. The finalconcentration of kerateines in these purified solutions can be adjustedby the addition/removal of water.

Regardless of the form of the keratin (i.e. keratoses or kerateines),several different approaches to further purification can be employed tokeratin solutions. Care must be taken, however, to choose techniquesthat lend themselves to keratin's unique solubility characteristics. Oneof the most simple separation technologies is isoelectric precipitation.In this method, proteins of differing isoelectric point can be isolatedby adjusting the pH of the solution and removing the precipitatedmaterial. In the case of keratins, both gamma- and alpha-forms aresoluble at pH>6.0. As the pH falls below 6, however, alpha-keratinsbegin to precipitate. Keratin fractions can be isolated by stopping theprecipitation at a given pH and separating the precipitate bycentrifugation and/or filtration. At a pH of approximately 4.2,essentially all of the alpha-keratin will have been precipitated. Theseseparate fractions can be re-dissolved in water at neutral pH, dialyzed,concentrated, and reduced to powders by lyophilization or spray drying.However, kerateine fractions must be stored in the absence of oxygen orin dilute solution to avoid crosslinking.

Another general method for separating keratins is by chromatography.Several types of chromatography can be employed to fractionate keratinsolutions including size exclusion or gel filtration chromatography,affinity chromatography, isoelectric focusing, gel electrophoresis, ionexchange chromatography, and immunoaffinity chromatography. Thesetechniques are well known in the art and are capable of separatingcompounds, including proteins, by the characteristics of molecularweight, chemical functionality, isoelectric point, charge, orinteractions with specific antibodies, and can be used alone or in anycombination to effect high degrees of separation and resulting purity.

A preferred purification method is ion exchange (IEx) chromatography.IEx chromatography is particularly suited to protein separation owningto the amphiphilic nature of proteins in general and keratins inparticular. Depending on the starting pH of the solution, and thedesired fraction slated for retention, either cationic or anionic IEx(CIEx or AIEx, respectively) techniques can be used. For example, at apH of 6 and above, both gamma- and alpha-keratins are soluble and abovetheir isoelectric points. As such, they are anionic and can be bound toan anionic exchange resin. However, it has been discovered that asub-fraction of keratins does not bind to a weakly anionic exchangeresin and instead passes through a column packed with such resin. Apreferred solution for AIEx chromatography is purified or fractionatedkeratin, isolated as described previously, in purified water at aconcentration between 0 and 5 weight/volume %. A preferred concentrationis between 0 and 4 w/v %. A most preferred concentration isapproximately 2 w/v %. It is preferred to keep the ionic strength ofsaid solution initially quite low to facilitate binding to the AIExcolumn. This is achieved by using a minimal amount of acid to titrate apurified water solution of the keratin to between pH 6 and 7. A mostpreferred pH is 6. This solution can be loaded onto an AIEx column suchas DEAE-Sepharose® resin or Q-Sepharose® resin columns. A preferredcolumn resin is DEAE-Sepharose® resin. The solution that passes throughthe column can be collected and further processed as describedpreviously to isolate a fraction of acidic keratin powder.

In some embodiments the activity of the keratin matrix is enhanced byusing an AIEx column to produce the keratin to thereby promote celladhesion. Without wishing to be bound to any particular theory, it isenvisioned that the fraction that passes through an anionic column, i.e.acidic keratin, promotes cell adhesion.

Another fraction binds readily, and can be washed off the column usingsalting techniques known in the art. A preferred elution medium issodium chloride solution. A preferred concentration of sodium chlorideis between 0.1 and 2M. A most preferred concentration is 2M. The pH ofthe solution is preferred to be between 6 and 12. A most preferred pH is12. In order to maintain stable pH during the elution process, a buffersalt can be added. A preferred buffer salt is Trizma® base. Thoseskilled in the art will recognize that slight modifications to the saltconcentration and pH can be made to effect the elution of keratinfractions with differing properties. It is also possible to usedifferent salt concentrations and pH's in sequence, or employ the use ofsalt and/or pH gradients to produce different fractions. Regardless ofthe approach taken, however, the column eluent can be collected andfurther processed as described previously to isolate fractions of basickeratin powders.

A complimentary procedure is also feasible using CIEx techniques.Namely, the keratin solution can be added to a cation exchange resinsuch as SP Sepharose® resin (strongly cationic) or CM Sepharose® resin(weakly cationic), and the basic fraction collected with the passthrough. The retained acid keratin fraction can be isolated by saltingas previously described.

Meta Keratins. Meta keratins are synthesized from both the alpha andgamma fractions of kerateine using substantially the same procedures.Basically, the kerateine is dissolved in a denaturing solution such as7M urea, aqueous ammonium hydroxide solution, or 20 mM tris buffersolution. Pure oxygen is bubbled through the solution to initiateoxidative coupling reactions of cysteine groups. The progress of thereaction is monitored by an increase in molecular weight as measuredusing SDS-PAGE. Oxygen is continually bubbled through the reactionsolution until a doubling or tripling of molecular weight is achieved.The pH of the denaturing solution can be adjusted to neutrality to avoidhydrolysis of the proteins by addition of mineral acid.

Keratin Intermediate Filaments. IFs of human hair fibers are obtainedusing the method of Thomas and coworkers (H. Thomas et al., Int. J.Biol. Macromol. 8, 258-64 (1986)). This is essentially a chemicaletching method that reacts away the keratin matrix that serves to “glue”the IFs in place, thereby leaving the IFs behind. In a typicalextraction process, swelling of the cuticle and sulfitolysis of matrixproteins is achieved using 0.2M Na₂SO₃, 0.1M Na₂O₆S₄ in 8M urea and 0.1Mtris-HCl buffer at pH 9. The extraction proceeds at room temperature for24 hours. After concentrating, the dissolved matrix keratins and IFs areprecipitated by addition of zinc acetate solution to a pH ofapproximately 6. The IFs are then separated from the matrix keratins bydialysis against 0.05M tetraborate solution. Increased purity isobtained by precipitating the dialyzed solution with zinc acetate,redissolving the IFs in sodium citrate, dialyzing against distilledwater, and then freeze drying the sample.

Further discussion of keratin preparations are found in U.S. PatentApplication Publication 2006/0051732 (Van Dyke), which is incorporatedby reference herein.

Compositions and formulations. Dry powders may be formed of keratinderivatives as described above in accordance with known techniques suchas freeze drying (lyophilization). In some embodiments, compositions ofthe invention may be produced by mixing such a dry powder compositionform with an aqueous solution to produce a composition comprising anelectrolyte solution having said keratin derivative solubilized therein.The mixing step can be carried out at any suitable temperature,typically room temperature, and can be carried out by any suitabletechnique such as stirring, shaking, agitation, etc. The salts and otherconstituent ingredients of the electrolyte solution (e.g., allingredients except the keratin derivative and the water) may becontained entirely in the dry powder, entirely within the aqueouscomposition, or may be distributed between the dry powder and theaqueous composition. For example, in some embodiments, at least aportion of the constituents of the electrolyte solution are contained inthe dry powder.

The formation of a matrix comprising keratin materials such as describedabove can be carried out in accordance with techniques long establishedin the field or variations thereof that will be apparent to thoseskilled in the art. In some embodiments, the keratin preparation isdried and rehydrated prior to use. See, e.g., U.S. Pat. No. 2,413,983 toLustig et al., U.S. Pat. No. 2,236,921 to Schollkipf et al., and U.S.Pat. No. 3,464,825 to Anker. In preferred embodiments, the matrix, orhydrogel, is formed by re-hydration of the lyophilized material with asuitable solvent, such as water or phosphate buffered saline (PBS). Thegel can be sterilized, e.g., by γ-irradiation (800 krad) using a Co60source. Other suitable methods of forming keratin matrices include, butare not limited to, those found in U.S. Pat. No. 6,270,793 (Van Dyke etal.), U.S. Pat. No. 6,274,155 (Van Dyke et al.), U.S. Pat. No. 6,316,598(Van Dyke et al.), U.S. Pat. No. 6,461,628 (Blanchard et al.), U.S. Pat.No. 6,544,548 (Siller-Jackson et al.), and U.S. Pat. No. 7,01,987 (VanDyke).

In some composition embodiments, the keratin derivatives (particularlyalpha and/or gamma kerateine and alpha and/or gamma keratose) have anaverage molecular weight of from about 10 to 70 or 85 or 100kiloDaltons. Other keratin derivatives, particularly meta-keratins, mayhave higher average molecular weights, e.g., up to 200 or 300kiloDaltons. In general, the keratin derivative (this term includingcombinations of derivatives) may be included in the composition in anamount of from about 0.1, 0.5 or 1 percent by weight up to 3, 4, 5, or10 percent by weight. The composition when mixed preferably has aviscosity of about 1 or 1.5 to 4, 8, 10 or 20 centipoise. Viscosity atany concentration can be modulated by changing the ratio of alpha togamma keratose.

The keratin derivative composition or formulation may optionally containone or more active ingredients such as one or more growth factors,analgesics, antimicrobials, additional coagulants, etc. (e.g., in anamount ranging from 0.0000001 to 1 or 5 percent by weight of thecomposition that comprises the keratin derivative(s)), to facilitategrowth or healing, provide pain relief, inhibit the growth of microbessuch as bacteria, facilitate or inhibit coagulation, facilitate orinhibit cell or tissue adhesion, etc. Examples of suitable growthfactors include, but are not limited to, nerve growth factor, vascularendothelial growth factor, fibronectin, fibrin, laminin, acidic andbasic fibroblast growth factors, testosterone, ganglioside GM-1,catalase, insulin-like growth factor-I (IGF-I), platelet-derived growthfactor (PDGF), neuronal growth factor galectin-1, and combinationsthereof. See, e.g., U.S. Pat. No. 6,506,727 to Hansson et al. and U.S.Pat. No. 6,890,531 to Horie et al.

As used herein, “growth factors” include molecules that promote theregeneration, growth and survival of tissue. Growth factors that areused in some embodiments of the present invention may be those naturallyfound in keratin extracts, or may be in the form of an additive, addedto the keratin extracts or formed keratin matrices. Examples of growthfactors include, but are not limited to, nerve growth factor (NGF) andother neurotrophins, platelet-derived growth factor (PDGF),erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growthdifferentiation factor-9 (GDF9), basic fibroblast growth factor (bFGF orFGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF),granulocyte-colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF). There aremany structurally and evolutionarily related proteins that make up largefamilies of growth factors, and there are numerous growth factorfamilies, e.g., the neurotrophins (NGF, BDNF, and NT3). Theneurotrophins are a family of molecules that promote the growth andsurvival of, inter alia, nervous tissue. Examples of neurotrophinsinclude, but are not limited to, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), andneurotrophin 4 (NT-4). See U.S. Pat. No. 5,843,914 to Johnson, Jr. etal.; U.S. Pat. No. 5,488,099 to Persson et al.; U.S. Pat. No. 5,438,121to Barde et al.; U.S. Pat. No. 5,235,043 to Collins et al.; and U.S.Pat. No. 6,005,081 to Burton et al.

For example, a growth factor can be added to the keratin matrixcomposition in an amount effective to promote the regeneration, growthand survival of various tissues. The growth factor is provided inconcentrations ranging from 0.1 ng/mL to 1000 ng/mL. More preferably,growth factor is provided in concentrations ranging from 1 ng/mL to 100ng/mL, and most preferably 10 ng/mL to 100 ng/mL. See U.S. Pat. No.6,063,757 to Urso.

The composition is preferably sterile and non-pryogenic. The compositionmay be provided preformed and aseptically packaged in a suitablecontainer, such as a flexible polymeric bag or bottle, or a foilcontainer, or may be provided as a kit of sterile dry powder in onecontainer and sterile aqueous solution in a separate container formixing just prior to use. When provided pre-formed and packaged in asterile container, the composition preferably has a shelf life of atleast 4 or 6 months (up to 2 or 3 years or more) at room temperature,prior to substantial loss of viscosity (e.g., more than 10 or 20percent) and/or substantial precipitation of the keratin derivative(e.g., settling detectable upon visual inspection). The kit may containa single unit dose of the active keratin derivative. A single unit dosemay be 0.1 or 0.5 or 1, to 100 or 200 or 300 grams of the keratinderivative, or more, depending upon its intended use.

Other examples of natural polymers that may be utilized in a similarfashion to the disclosed keratin preparations include, but are notlimited to, collagen, gelatin, fibronectin, vitronectin, laminin,fibrin, mucin, elastin, nidogen (entactin), proteoglycans, etc. (See,e.g., U.S. Pat. No. 5,691,203 to Katsuen et al.).

Clotting compositions and methods to control bleeding containing keratinbiomaterials. One aspect of the present invention is a method fortreating bleeding in a subject afflicted with a bleeding woundcomprising: applying a keratin derivative to a bleeding wound in anamount effective to treat the bleeding. In some embodiments, the keratinderivative comprises, consists of or consists essentially of kerateine,alpha kerateine, gamma kerateine, acidic alpha kerateine, basic alphakerateine, or combinations thereof, such as described above. Thebleeding may be that associated with, e.g., severe trauma producingrapid, voluminous hemorrhaging, including, but not limited to: surgery;penetrating trauma such as stabbing and gunshot wounds; motor vehicletrauma; and head, neck, chest and abdominal hemorrhaging; with orwithout clear access to the site of the hemorrhaging.

Many different compositions may comprise the hemostatic agent,including, but not limited to, keratin derivatives. Other examples ofhemostatic agents include, but are not limited to, those comprisingfibrin or fibrinogen, thrombin, factor XIII, calcium, chitosan(deacetylated poly-N-acetyl glucosamine), zeolite (oxides of silicon,aluminum, sodium, magnesium, and quartz), chitin (acetylatedpoly-N-acetyl glucosamine), bovine clotting factors, non-zeolite mineral(e.g., hydrophobic polymers and potassium salts), and molecular sievematerials from plant sources (e.g. TraumaDEX™, Arista™ AH, etc.,Medafor, Inc., Minneapolis, Minn.). It should be noted, however, thatnot all of these hemostatic agents are recommended for all types ofbleeding treatments, and those skilled in the art should selecthemostatic agents for use in the disclosed compositions and methodsaccordingly. For example, zeolite is intended only for external use.

Wound healing compositions containing keratin biomaterials and methodsto promote healing. Another aspect of the present invention is a methodof treating a wound (e.g., burns, abrasions, incisions, pressure sores,etc.) in a subject in need thereof, comprising: topically applying akeratin derivative to the wound in an amount effective to treat thewound. In some embodiments the keratin derivative comprises, consists ofor consists essentially of keratose, alpha keratose, acidic alphakeratose, kerateine, alpha kerateine, acidic alpha kerateine, etc., orcombinations thereof, such as described above. The keratin derivativecan be topically applied as a dry powder formulation or, in someembodiments, applied in an aqueous carrier.

The dose of the keratin material depends upon the particular disorder orwound, age and condition of the subject, route of administration, etc.,and can be optimized in accordance with known techniques. In someembodiments, the dosage will be dose may be 0.1 or 0.5 or 1, to 100 or200 or 300 grams of the keratin derivative, or more, depending upon itsintended use.

Surgical or paramedic aids. Another aspect of the invention is asurgical or paramedic aid, comprising a solid, physiologicallyacceptable substrate and a keratin derivative on said substrate.“Substrate” includes sponges, packings, wound dressings (such as gauzeor bandages), sutures, fabrics, and prosthetic devices.

Embodiments of the invention are further described in the followingnon-limiting examples.

EXAMPLE 1 Keratin Derivatives/Fractions

Keratose fractions were obtained using a method based on that ofAlexander and coworkers. However the method was substantially modifiedto minimize hydrolysis of peptide bonds. Briefly, 50 grams of clean, dryhair that was collected from a local barber shop was reacted with 1000mL of an aqueous solution of 2 w/v % peracetic acid (PAA) at roomtemperature for 12 hr. The oxidized hair was recovered using a 500micron sieve, rinsed with copious amounts of DI water, and the excesswater removed. Keratoses were extracted from the oxidized hair fiberswith 1000 mL of 100 mM Trizma® base. After 3 hours, the hair wasseparated by sieve and the liquid neutralized by dropwise addition ofhydrochloric acid (HCl). Additional keratoses were extracted from theremaining hair with two subsequent extractions using 1000 mL of 0.1MTrizma® base and 1000 mL of DI water, respectively. Each time the hairwas separated by sieve and the liquid neutralized with HCl. All threeextracts were combined, centrifuged, and any residual solid materialremoved by filtration. The combined extract was purified by tangentialflow dialysis against DI water with a 1 KDa nominal low molecular weightcutoff membrane. The solution was concentrated and lyophilized toproduce a crude keratose powder.

Kerateine fractions were obtained using a modification of the methoddescribed by Goddard and Michaelis. Briefly, the hair was reacted withan aqueous solution of 1M TGA at 37° C. for 24 hours. The pH of the TGAsolution had been adjusted to pH 10.2 by dropwise addition of saturatedNaOH solution. The extract solution was filtered to remove the reducedhair fibers and retained. Additional keratin was extracted from thefibers by sequential extractions with 1000 mL of 100 mM TGA at pH 10.2for 24 hours, 1000 mL of 10 mM TGA at pH 10.2 for 24 hours, and DI waterat pH 10.2 for 24 hours. After each extraction, the solution wascentrifuged, filtered, and added to the dialysis system. Eventually, allthe extracts were combined and dialyzed against DI water with a 1 KDanominal low molecular weight cutoff membrane. The solution wasconcentrated, titrated to pH 7, and stored at approximately 5% totalprotein concentration at 4° C. Alternately, the concentrated solutioncould be lyophilized and stored frozen and under nitrogen.

Just prior to fractionation, keratose samples were re-dissolved inultrapure water and titrated to pH 6 by addition of dilute HCl solution.Kerateine samples were titrated to pH 6 by careful addition of diluteHCl solution as well. The samples were loaded onto a 200 mL flashchromatography column containing either DEAE-Sepharose (weakly anionic)or Q-Sepharose (strongly anionic) exchange resin (50-100 mesh;Sigma-Aldrich, Milwaukee, Wis.) with gentle pressure and the flowthrough collected (acidic keratin). A small volume of 10 mM Trizma® base(approximately 200 mL) at pH 6 was used to completely wash through thesample. Basic keratin was eluted from the column with 100 mM tris baseplus 2M NaCl at pH 12. Each sample was separately neutralized anddialyzed against DI water using tangential flow dialysis with a LMWCO of1 KDa, concentrated by rotary evaporation, and freeze dried.

As previously described, a sample of alpha-keratose was produced,separated on a DEAE-Sepharose IEx column into acidic and basicfractions, dissolved in PBS, and the pH adjusted to 7.4. These solutionswere prepared at 5 weight percent concentration and their RBCaggregation characteristics grossly evaluated with fresh whole humanblood by mixing at a 1:1 ratio. Samples were taken after 20 minutes andevaluated by light microscopy. The ion exchange chromatography washighly effective at separating the aggregation phenomenon (data notshown). Basic alpha-keratose was essentially free from interactions withblood cells, while the acidic alpha-keratose caused excessiveaggregation.

Samples of acidic and basic alpha-keratose, unfractionatedalpha+gamma-kerateines, unfractionated alpha+gamma-keratose, andbeta-keratose (derived from cuticle) were prepared at approximately 4w/v % and pH 7.4 in phosphate buffered saline (PBS). Samples were testedfor viscosity and red blood cell (RBC) aggregation. These results areshown in Table 1:

TABLE 1 Results of viscosity and RBC aggregation tests on keratinsolutions. Fluid formulations were prepared at approximately 4 w/v % inPBS at pH 7.4 and tested with human whole blood at a ratio of 1:1.Viscosity RBC Sample Description (centipoise) Aggregation* acidicalpha-keratose (1X AIEx) 5.65 3 acidic alpha-keratose (2X AIEx) 19.7 5basic alpha-keratose 1.57 2 alpha + gamma-keratose (hydrolyzed) 1.12 1alpha + gamma-kerateine (unfractionated) 1.59 2 *Degree of aggregation:1 = none, 5 = high

EXAMPLE 2 Animal Model

The hemostatic potential of keratin gel was evaluated in a modestlychallenging animal model. The keratin gel comprised unfractionatedkerateine (alpha+gamma). Liver injuries are notoriously problematic asboth the size of the liver and of the wound increase. This rabbit modelcan produce both profuse and lethal hemorrhage. Controlled livertransection was used as a means to establish a consistent set ofconditions that would result in exsanguination in the absence oftreatment (negative control), yet provide for the recovery of testanimals when a conventional hemostat was applied (positive control). Itshould be noted that the hemostats used as positive controls in thisstudy are indicated for topical wounds and require concomitant pressure;they were applied without compression in this study. This was done toavoid the confounding contribution compression would add as it was notused with the keratin gel.

A total of 16 New Zealand rabbits (3.7 kg average) were used in thisstudy. The animals received a standardized liver injury that consistedof transection of approximately one third of the left central lobe andwere then randomized into one of four groups. Four animals served asnegative controls and received no treatment, four animals receivedtreatment with QuikClot® hemostatic agent, four animals were treatedwith HemCon® hemostatic bandage, and four animals were treated withkeratin gel. No resuscitation fluids were given and all animals wereclosely monitored during surgery. After one hour the surgical wound wasclosed and the animals transferred to the housing facility. Allsurviving animals were sacrificed after 72 hours. At the time ofsacrifice, liver tissue was retrieved for histological analysis.

EXAMPLE 3 Surgeries and Postoperative Treatment

All procedures were performed in accordance with Wake ForestUniversity's Animal Care and Use Committee guidelines, which encompassregulatory and accreditation agencies' guidelines. The animals wereweighed immediately before surgery. All animals were sedated using acombination of Ketamine 10 mg/kg and Xylazine 4 mg/kg through anintramuscular injection, intubated and maintained on 2-3% Isoflurane forthe remainder of the procedure. The animals were then placed in a supineposition, shaved and connected to the monitoring devices. All animalswere connected to ECG leads, pulse oximeter cuff on the tail, and anintra-esophageal probe for temperature monitoring. After sterileprepping and draping, the abdominal incision was performed and the liverexposed. Prior to the liver injury, the abdominal aorta of the animalswas exposed and canulated using a 23 gauge needle connected to apressure transducer (Lab-stat, ADInstruments Pty. Ltd. Castle Hill,Australia) which in turn was connected to a PowerLab® (ADInstruments)system for data acquisition. The mean arterial pressure (MAP) wasrecorded continuously throughout the procedure. All animals weremonitored for several minutes and assured to be in a stable state priorto liver injury. The median lobe of the liver was used for the injurydue to its ample size and easy accessibility.

Preliminary data during model development showed that a consistent liverinjury cross sectional area could be created that resulted in death whenleft untreated, but that when treated with a control material couldrescue the animal. A 2.0 cm² surface area ring was used to inflict aconsistent sized injury to the liver by pulling the left central lobethrough the ring and cutting immediately adjacent to the ring with asurgical blade. The MAP, temperature, heart rate, O₂ saturation, andshed blood were recorded throughout the procedure at 30 seconds, 5, 15,30, 45 and 60 minutes. Shed blood was measured at each time point usingpre-weighed sterile surgical gauze which was placed under the liverinjury. In addition, blood samples were taken for CBC through an earvein.

All animals were randomized into the previously mentioned fourexperimental groups. The negative control group did not receive anytreatment and the time of death was recorded in minutes after inflictionof the injury. As for the other experimental groups, the treatment wasadministered at the 5 minute time point unless the MAP fell to half ofthe starting value. For standardization, the hemostatic materialsapplied were measured or weighed. The keratin gel does not requirecompression so no compression was used in any of the other treatmentgroups so as not to confound the results. In the HemCon® hemostaticbandage treatment group, a 4.5×2.5 cm piece of bandage that was placedon the bleeding surface of the liver throughout the procedure and wasremoved prior to closure. In the QuikClot® hemostatic agent treatedgroup, 2.5 grams of autoclave sterilized material per animal was used.The material was spread on the bleeding surface and was left afterclosure in the surviving animals. In the case of the keratin treatmentgroup, 2 ml of the gel was used per animal. Sterile keratin gel wasapplied to the bleeding surface through a 1 ml syringe.

The keratin was also left in place after closure of the animals. Theseparameters were determined during initial model development based oncomplete coverage of the wound site. For the surviving animals, themonitoring continued for 60 minutes, after which the animal wasconsidered to have survived the initial trauma and the bleeding stopped.The animals that were treated with HemCon® hemostatic bandage had toundergo removal of the material since it could not be leftintraabdominally as indicated by the manufacturer. The aortic cannulawas removed and hemostasis established at the insertion site. No aorticbleeding was observed in any animal at necropsy. The fascia and skin ofthe abdomen was closed in two layers. After complete closure of theabdomen, the animals were allowed to recover and transported to thehousing facility where they were monitored every 15 minutes untilcomplete recovery from anesthesia, then three times per day thereafterfor the following three days. Blood samples were taken from allsurviving animals every day for CBC analysis. Upon sacrifice at the 72hour time point, the liver of each animal was harvested for histologicalevaluation.

All presented data is expressed as averages and the correspondingstandard deviations. For statistical analysis, SPSS v.11 (SPSS Inc,Chicago, Ill.) was used. Outliers were defined as having a z-scorelarger then +3.0 or smaller then −3.0 using a modified z-score (medianof the absolute deviation). Data at all time points were analyzed byone-way analysis of variance (ANOVA). If significant F values werefound, the groups were further analyzed by Fischer's Least SignificantDifference Test (LSD). An alpha of p<0.05 was considered significant.The probability of a Type I error was minimized by limiting comparisons;only negative control versus the 3 treatment groups were performed. Inorder to compensate for bias generated by early drop out of dead animals(i.e. animals that exsanguinated before the end of the 60 minuteoperative period), polynomial regression to known pathologic endpointswas used to estimate values during the first 60 minutes. For the percentblood loss graphical data (FIG. 2) where statistical relevance wasreached with some groups, values are expressed as means with theircorresponding standard error.

Negative control animals (i.e. no treatment), as expected, exsanguinatedwithin the 60 minute operative period (31±19 minutes). Two animals inthe QuikClot® hemostatic agent group and one in the HemCon® hemostaticbandage group did not survive beyond the initial 60 minute operativeperiod. Also in the HemCon® hemostatic bandage group, one animal waseuthanized 24 hours post-op on the advice of the veterinary staff. Thisanimal was not ambulatory and could not eat or drink. One animal in thekeratin group was also sacrificed at 48 hours. Although the animal wasmoving freely in its cage, it was not eating or drinking. At necropsy,these animals showed no evidence of additional bleeding after theoperative period. All other surviving animals recovered withoutincident, were freely moving in their cages within 24 hours, and hadnormal CBC by 72 hours (data not shown). A summary of the survival datais shown in FIG. 1.

Mean Arterial Pressure. The mean arterial pressure (MAP) was recordedusing a 23 gauge needle placed into the lower part of the abdominalaorta. The needle was connected to a PE 50 tube, which in turn wasconnected to a pressure transducer (Lab-stat) that was connected to aPowerLab system for pressure recording. The MAP was continuouslymonitored during the entire course of the procedure or until the deathof the animal. To further evaluate the significance of a change in MAPand heart rate, shock index was used. Shock index is a well establishedclinical scoring system for fast assessment of trauma patients. Themodified shock index was calculated by dividing heart rate by MAP(mmHg).

The mean arterial pressure in the abdominal aorta was recorded for 60minutes. Animals in the keratin and HemCon® hemostatic bandage groupwere able to achieve stable MAPs after 5 minutes at 75% of the startingvalue. The QuikClot® hemostatic agent and control groups failed tostabilize MAP and dropped to 45% of the starting value after 60 minutes(FIG. 3). However, these data did not reach statistical significancebetween groups.

The shock index (SI), a predictive score grading system for the severityof blood loss, showed a beneficial outcome for the keratin group withlow values throughout the first 60 minutes (FIG. 4). The high values ofQuikClot® hemostatic agent matched with two early deaths during thefirst 20 minutes of observation supporting the predictive nature of thismeasure. Although a trend was noted, these data did not reachstatistical significance in the present study.

Temperature, ECG and Heart Rate. The central temperature was recordedwith an esophageal probe connected to the surgery room monitor. Thetemperature of the animal was continuously monitored throughout theprocedure and recorded at the previously mentioned time points. The ECGand heart rate were monitored using a three lead system connected to thesurgery room monitor and was maintained throughout the entire procedure.Flat line or irregular electrical activity with electrical mechanicaldissociation was used to define the time of death.

The liver damage model employed in this study represented severe traumawith significant, rapid blood loss. The liver transection produced alethal injury, typically involving one or two large vessels ofapproximately 1 mm diameter and several in the 0.5 to 1.0 mm diameterranges. The severity of the injury was such that untreated rabbits allexsanguinated within the 60 minute operative period. None of the animalswere able to compensate for loss of blood volume with an increase inheart rate. All animals showed a comparable decrease from 263 bpm to 188bpm after 30 minutes and 154 bpm after one hour. There were nostatistically significance differences between the groups. However, thekeratin group showed a trend toward compensation and recovery with anincrease in heart rate in the second half of the surgical period from 30min to 60 min. The temperature of all animals dropped in a similarfashion with a step drop of 0.8° C. in the first 5 minutes and a totalof 2.7° C. over 60 minutes. There was no statistically significantdifference between the experimental groups.

Shed Blood. Shed blood was measured by weight after subtracting theweight of the pre-weighed gauze. Weights were recorded at each timepoint and fresh gauze placed under the liver injury. The shed blood wasrepresented as a percent of the original body weight for each animal.CBC was determined from samples taken from an ear vein on a HEMAVet®multi-species hematology system (Model 950FS, Drew Scientific, Dallas,Tex.).

Blood loss was measured by weighing the surgical gauze placed below theinjured liver lobe. The blood loss was expressed as percentage ofstarting body weight. As expected in uncontrolled hemorrhage studies,all animals showed an initial phase of profuse bleeding followed by alinear phase with a lower bleeding rate, as MAP falls (FIG. 2). Acomparison of the keratin and QuikClot® hemostatic agent groups to thenegative controls shows a significantly decreased amount of blood lossat the 30, 45, and 60 minute time points (p values for keratin vs.negative control were 0.018, 0.011 and 0.007; p values for QuikClot®hemostatic agent vs. negative control were 0.009, 0.005 and 0.004,respectively).

As one would expect, the survivability of the animals appeared to bedependent on the vascular anatomy at the injury site, which was notconsistent from animal to animal even though the total surface areatransected was controlled. When a single very large bleeder (>1 mm), ormultiple large bleeders (>2 to 3 in the 1 m size range) were encounteredwithin the injury area, the animal's chance of survival was negligiblein the QuikClot® hemostatic agent and HemCon® hemostatic bandage groups.In the QuikClot® hemostatic agent group in particular, a single verylarge bleeder or an excess of 2 to 3 large bleeders would ensurelethality. It should be noted however, that when used according tomanufacturer's instructions with concomitant pressure, other studieshave shown better survival rates using QuikClot® hemostatic agent andHemCon® hemostatic bandage. In all cases of treatment with keratin gel,which was also used without any compression, the animals survived for atleast 24 hours, regardless of the size of the severed vessels. Althougha small number of animals were used in all test groups (n=4), theseoutcomes are encouraging.

The keratin hemostatic gel consistently performed well by each outcomemeasure, particularly shed blood volume, MAP, and (importantly)survival. One particularly distinguishing outcome was shock index. Inmost cases of hemorrhage, cardiac output is increased to compensate forthe drop in blood pressure. Once this mechanism takes over, the value ofshock index increases rapidly and survivability becomes doubtful.Remarkably, the shock index in the keratin treatment group remained thelowest of all the materials tested, consistent with early effectivehemostasis.

EXAMPLE 4 Histology

A tissue sample including the damaged liver surface was removed fromeach animal within one hour of euthanasia. Each sample was placed inTissue-Tek® O.C.T. Compound 4583 (Sakura®) and frozen in liquidnitrogen. The frozen blocks were sectioned into 8 μm slices using acryostat (Model CM 1850, Leica Microsystems, Bannockburn, Ill.) toinclude the transected portion of the liver and mounted onto microscopeslides. The slides were fixed and stained with Hematoxylin and Eosin(H&E). Technical difficulties in sectioning arose with both theQuikClot® hemostatic agent and the HemCon® hemostatic bandage sections.The brittle QuikClot® hemostatic agent made level sectioning difficultand created voids in the sections. The HemCon® hemostatic bandagebandage was removed before abdominal closure and therefore the clottedblood was only partially visible. Digital images were taken (Zeiss AxioImager M1 Microscope, Carl Zeiss, Thornwood, N.Y.) at varyingmagnifications to observe the interactions between the hemostat and thedamaged area of the liver. A magnification of 100× showed the overallresponse of the tissue, while magnifications of 200× and 400× were usedto visualize the cellular response.

The transected liver surfaces were examined by light microscopy of H&Estained sections. The negative control group showed a clean cut with notissue response or necrosis (FIG. 5A). Moreover, no functional clottingwas observed with little thrombus adhered to the surface. The QuikClot®hemostatic agent samples were difficult to process due to the presenceof this hard, granular zeolite in the clot. Histology revealed necrotictissue mixed with blood clots (FIG. 5B). The transparent areas representQuikClot® hemostatic agent particles removed during processing. TheHemCon® hemostatic bandage group showed some areas with clotted bloodand adjacent cellular infiltration (FIG. 5C). Since the HemCon®hemostatic bandage was removed after 60 minutes, most of the liversurfaces had only a thin layer of blood clots. The keratin group showeda thick layer of biomaterial attached to the damaged liver surface (FIG.5D). Granulation-like tissue with cellular infiltration had formed inthe pores of the keratin biomaterial gel (FIG. 6).

The keratin hemostatic gel was adherent to the tissue and hydrophilic.When deposited onto the bleeding surface of the liver it wassufficiently adhesive to not be washed away, even in the presence ofprofuse bleeding. The gel absorbed fluid from the blood and became evenmore adherent within a few minutes of administration. Clotting andadherence was almost instantaneous with contact. Interestingly, thekeratin gel formed a thick seal of granulation-like tissue over thewound site by 72 hours. Upon inspection of histological sections, 3 daysafter injury host cells could be seen infiltrating the gel. It isbelieved that the keratin gel used in these experiments serves twopurposes. First, contact of the gel with whole blood instigates thrombusformation, probably through platelet activation or concentration ofclotting factors. Second, the adherent gel forms a physical seal of thewound site and provides a porous scaffold for cell infiltration andgranulation-like tissue formation, much like clotted blood.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A pharmaceutical composition comprising: a) a keratin derivative; and b) optionally, at least one additional active ingredient; wherein, said keratin derivative comprises keratose, kerateine, or a combination thereof.
 2. The composition of claim 1, wherein said keratin derivative comprises kerateine.
 3. The composition of claim 1, wherein said keratin derivative consists essentially of keratose, kerateine, or a combination thereof.
 4. The composition of claim 1, wherein said keratin derivative consists essentially of kerateine.
 5. The composition of claim 1, wherein said keratin derivative consists essentially of acidic kerateine.
 6. The composition of claim 1, wherein said keratin derivative consists essentially of acidic alpha kerateine.
 7. The composition of claim 1, wherein said keratin derivative consists essentially of acidic gamma kerateine.
 8. The composition of claim 1, wherein said at least one additional active ingredient is selected from the group consisting of analgesics, antimicrobial agents, and additional coagulants.
 9. The composition of claim 1, wherein said additional active ingredient is a coagulant is selected from the group consisting of zeolite-based coagulant and chitosan-based coagulant.
 10. A method for treating bleeding in a subject afflicted with a bleeding wound comprising applying a positively charged composition to said wound in an amount effective to treat said bleeding.
 11. The method of claim 10, wherein said positively charged composition is selected from the group consisting of derivatives comprising: keratin, collagen, mucin, elastin, gelatin, fibronectin, vitronectin and laminin.
 12. A method for treating bleeding in a subject afflicted with a bleeding wound, comprising: applying a keratin derivative to said wound in an amount effective to treat said bleeding; wherein said keratin derivative consists essentially of keratose, kerateine, or combinations thereof.
 13. The method of claim 12, wherein said keratin derivative consists essentially of a kerateine.
 14. The method of claim 12, wherein said keratin derivative consists essentially of an alpha kerateine.
 15. The method of claim 12, wherein said keratin derivative consists essentially of acidic alpha kerateine.
 16. The method of claim 15, wherein said acidic alpha kerateine is produced by the process of fractionating a mixture comprising acidic and basic alpha kerateine by ion exchange chromatography.
 17. The method of claim 12, wherein said keratin derivative consists essentially of basic alpha kerateine.
 18. The method of claim 17, wherein said basic alpha kerateine is produced by the process of fractionating a mixture comprising acidic and basic alpha kerateine by ion exchange chromatography.
 19. A method of treating a wound in a subject in need thereof, comprising: topically applying a keratin derivative to said wound in an amount effective to treat said wound, wherein said keratin derivative consists essentially of a keratose, a kerateine, or combinations thereof.
 20. The method of claim 19, wherein said keratin derivative consists essentially of a kerateine.
 21. The method of claim 19, wherein said keratin derivative consists essentially of an alpha kerateine.
 22. The method of claim 19, wherein said keratin derivative consists essentially of acidic alpha kerateine.
 23. The method of claim 22, wherein said acidic alpha kerateine is produced by the process of fractionating a mixture comprising acidic and basic alpha kerateine by ion exchange chromatography.
 24. The method of claim 19, wherein said keratin derivative consists essentially of a keratose.
 25. The method of claim 19, wherein said keratin derivative consists essentially of an alpha keratose.
 26. The method of claim 19, wherein said keratin derivative consists essentially of acidic alpha keratose.
 27. The method of claim 26, wherein said acidic alpha keratose is produced by the process of fractionating a mixture comprising acidic and basic alpha keratose by ion exchange chromatography.
 28. A surgical or paramedic aid, comprising: a solid, physiologically acceptable substrate; and a keratin derivative on said substrate wherein said keratin derivative consists essentially of a keratose, a kerateine, or combinations thereof.
 29. The surgical or paramedic aid according to claim 28, wherein said substrate is selected from the group consisting of sponges, packings, wound dressings, sutures, fabrics, and prosthetic devices.
 30. The surgical or paramedic aid according to claim 28, wherein said surgical or paramedic aid is sterile, and wherein said surgical or paramedic aid is packaged in a sterile container.
 31. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of a kerateine.
 32. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of an alpha kerateine.
 33. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of acidic alpha kerateine.
 34. The surgical or paramedic aid according to claim 33, wherein said acidic alpha kerateine is produced by the process of fractionating a mixture comprising acidic and basic alpha kerateine by ion exchange chromatography.
 35. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of a keratose.
 36. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of an alpha keratose.
 37. The surgical or paramedic aid according to claim 28, wherein said keratin derivative consists essentially of acidic alpha keratose.
 38. The surgical or paramedic aid according to claim 28, wherein said acidic alpha keratose is produced by the process of fractionating a mixture comprising acidic and basic alpha keratose by ion exchange chromatography.
 39. A kit comprising: a) a keratin derivative; and b) a container in which said keratin derivative is packaged in said container in sterile form, and wherein said keratin derivative comprises keratose, kerateine, or mixtures thereof.
 40. The kit of claim 39, wherein said keratin derivative is provided in hydrated or dehydrated form.
 41. The kit of claim 39, wherein said container comprises a foil container.
 42. The kit of claim 39, wherein said container is vacuum-packed.
 43. The kit of claim 39, wherein said keratin derivative comprises a single unit dose.
 44. The kit of claim 39, wherein said keratin derivative comprises 0.5 to 200 grams of dehydrated keratose, kerateine, or mixtures thereof.
 45. The kit of claim 39, wherein said keratin derivative comprises 0.5 to 200 milliliters of hydrated keratose, kerateine, or mixtures thereof.
 46. The kit of claim 39, further comprising a physiologically acceptable substrate.
 47. The kit of claim 46, wherein said substrate is sterile, and wherein said substrate is packaged in said container in sterile form.
 48. The kit of claim 46, wherein said substrate is selected from the group consisting of sponges, packings, wound dressings, sutures, fabrics, and prosthetic devices. 